Klebsiella pneumoniae厌氧发酵甘油生产1，3-丙二醇的过程研究
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摘要
1,3-丙二醇是一种重要的化工原料,可以用作聚酯和聚氨酯的合成单体,因其在纤维工业上的广泛应用而成为近年来研究的热点。目前1,3-丙二醇的生产主要是通过化学法,但是生物转化法因成本较低,可以利用甘油、葡萄糖等可再生资源等优点,受到越来越多的重视。
为了使生物转化法生产的1,3-丙二醇在市场上具有竞争力,需要提高1,3-丙二醇的浓度、得率和生产强度。本文对发酵过程中的培养基、通气条件、发酵动力学进行了研究。在此基础上,通过发酵过程中添加有机酸混合液和采取反复补料分批发酵的策略,显著提高了1,3-丙二醇的生产,具体实验结果如下：
利用正交实验,分别对种子培养基和发酵培养基进行了优化。在厌氧摇瓶中,利用优化后的发酵培养基,菌体生长和甘油的消耗显著提高,1,3-丙二醇的浓度和得率分别达到6.06 g·L-1和0.41 mol·mol-1,分别比在原始发酵培养基下提高了62%和7.9%。在5-L发酵罐中也得到类似的结果,并且优化后的发酵培养基中酵母提取物和氯化铵的浓度分别比原始发酵培养基显著下降,降低了生产的原料成本。
在原始发酵条件下,采取两阶段通气发酵及在发酵后期加入营养物质的策略不能提高1,3-丙二醇的生产。在250-mL摇瓶中,发现装液量增大时,有利于甘油的消耗和1,3-丙二醇的生成。在此基础上,分别研究了以甘油为唯一碳源情况下,不同通气方式对1,3-丙二醇生产的影响。结果显示,发酵过程中全程厌氧通气相比两阶段通气和通入空气,更有利于1,3-丙二醇的生产。
在补料分批发酵过程中,菌体的比生长速率随着产物的积累而不断的下降,进而影响到甘油的消耗和1,3-丙二醇的生成。在此基础上,通过甘油的限制性流加,控制菌体的比生长速率,发现在较低的菌体比生长速率下,甘油比消耗速率及其它产物的比生成速率较低,但是乙醇和甲酸的得率很高。随着菌体比生长速率的增加,甘油的比消耗速率和1,3-丙二醇的比生成速率迅速增加,同时乙醇和甲酸的得率迅速下降,1,3-丙二醇的得率大幅度增加。然后,在菌体生长和1,3-丙二醇生成明显减慢的发酵后期,通过控制不同的甘油浓度,发现甘油浓度在15-26 g.L-1时最有利于1,3-丙二醇的生成。最后,采取控制较高的菌体比生长速率和在发酵后期控制适宜甘油浓度相结合的策略,有效的减少了发酵过程中乳酸和乙醇的生成,提高1,3-丙二醇的得率。
在不对菌体进行基因工程操作的情况下,通过在发酵过程中外源添加有机酸混合液,有效的提高了1,3-丙二醇的生产,降低了乳酸和乙醇等副产物的生成。在此基础上,分别考察了不同浓度有机酸混合液加入时和在不同发酵时间加入时对发酵结果的影响。结果显示,在菌体浓度(OD650)达到3.0时,加入3.80 mM的有机酸混合液(柠檬酸、琥珀酸和延胡索酸均等浓度)最有利于1,3-丙二醇的生成,在30 h时达到了70 g.L-1以上。
在补料分批发酵过程中,产物积累到一定浓度时,就会对菌体生长产生很强的抑制。在厌氧摇瓶中通过反复分批培养,菌体生长和1,3-丙二醇的生产能力保持相对恒定；而在5-L发酵罐中发现,菌体长时间暴露在高浓度的产物中,不利于下一发酵循环的菌体生长。通过初始发酵条件下的反复补料分批发酵,1,3-丙二醇的生产强度为2.86g.L-1.h-1,但其浓度仅为44.60 g.L-1。在此基础上,采取反复补料分批发酵和有机酸添加相结合的策略,1,3-丙二醇生产有了显著的提高。在酵母浸膏代替酵母提取物的基础上,采取多循环的反复补料分批发酵操作,1,3-丙二醇的浓度、得率和生产强度在发酵20 h左右时分别达到66 g·L-1.0.61 mol·mol-1和3.40 g·L-1·h-1左右,显著的提高了1,3-丙二醇的生产。
1,3-propanediol has become one of the most interesting feedstocks in recent years because of its wide industrial applications, such as synthesis of polytrimethylene terephthalate and other polyester fibers. Current commercial production is based on the chemical route, which uses acrolein or ethylene as the starting material. Compared with the chemical synthesis method, biotransformation has the advantage of utilizing inexpensive, renewable resources such as glycerol or glucose, and becomes increasingly attractive.
To make the price more attractive, the yield, productivity and concentration of 1,3-propanediol need to be enhanced. In this paper, we had a study on culture medium optimization, aeration conditions and dynamics of fed-batch culture. Based on this, 1,3-propanediol production was enhanced by repeated fed-batch culture with organic acids addition, and the results of experiments were as follows.
The composition of medium for seeds culture and fermentation were optimized separately by orthogonal experiments. Based on this, the cell growth and glycerol consumption increased significantly in anaerobic bottle and the concentration and yield of 1,3-propanediol reached 6.06 g-L-'and 0.41 mol-mol-1, respectively,62% and 7.9% higher than that of the control, respectively. At the same time, the 1,3-propanediol production in 5-L bioreactor was also enhanced significantly, similar to that in anaerobic bottle. In addition, the concentrations of yeast extract and ammonium chloride both decreased sharply compared with that of the control, which could decrease the cost of the 1,3-propanediol production.
Under initial conditions, the 1,3-propanediol production would not be enhanced under the strategy of two-stage aeration and nutrient addition in late phase of fermentation. At the same time, experiments carried out in 250-mL with aerobic condition indicated that the increased volume of culture medium would be benefitial to PDO production. Based on this, effects of strategies including two-stagy aerating, aeration air and anaerobic aeration on the 1,3-propanediol production was studied separately as the glycerol was used as the solely carbon source, the results of experiments indicated that aerating N2 to maintain anaerobic condition would be benefitial to 1,3-propanediol production than that of others.
The specific growth rate of cells in fed-batch culture decreased as the products increasingly accumulated in culture medium, which further would not be benefitial to glycerol consumption and 1,3-propanediol production. Based on this, the specific growth rate was controlled by glycerol feeding rate. The experimental results indicated the specific growth rate affected significantly yields of productions. The yields of ethanol and formic acid on glycerol were high as the specific growth rate was maintained at low level. Increased the specific growth rate, the yields of ethanol and formic acid on glycerol decreased sharply while the yield of 1,3-propanediol on glycerol increased significantly. At the same time, the glycerol specific consumption rate and PDO formation rate both rapidly increased. As the cell growth and 1,3-propanediol specific formation rate declined sharply in late phase of fermentation, the glycerol concentration at the range of 15-26 g-L-1 was testified to be beneficial to the yield of 1,3-propanediol increase. Combined the specific growth rate controlled at higher level and glycerol concentration maintained range of 15-26 g-L-1 could effectively decreased the yields of lactic acid and ethanol on glycerol while increased the yield of 1,3-propanediol on glycerol.
The 1,3-propanediol production could be enhanced significantly while lactic acid and ethanol production decreased sharply with the parent strain under the strategy of organic acids mixture addition in the process of fermentation. Based on this, the effects of concentration of organic acids mixture addtion and time of organic acids mixture addition was studied separately and the results indicated that 3.80 mM organic acids mixture was added at the biomass (OD650) of 3.0 would be benefitial to 1,3-propanediol production and the concentration of 1,3-propanediol reached above 70 g-L"1at 30 h of culture.
The accumulation of products in medium of fed-batch culture would strongly inhibit cell growth. The strain used for 1,3-propanediol production could maintain the ability of 1,3-propanediol production in long-time cultivation in repeated batch culture in anaerobic bottle. However, when the cells used as inoculums which was maintained for a long-time in late phase of culture would not be benefitial to cell growth in next cycle of repeated fed-batch culture. The 1,3-propanediol productivity and concentration reached 2.86 g-L"--h-1and 44.60 g·LL1, respectively, in repeated fed-batch culture under initial condition. Based on this, the 1,3-propanediol production could be further enhanced in repeated fed-batch culture with organic acids mixture addition. At the same time, multiple-cycle of repeated fed-batch culture with industry-grade yeast extract replacing regent-grade yeast extract, the concentration, yield and productivity of 1,3-propanediol reached 66 g-L-,0.61 mol-mol1 and 3.40 g-L--h-, respectively, which was higher than that of the reported.
为了使生物转化法生产的1,3-丙二醇在市场上具有竞争力,需要提高1,3-丙二醇的浓度、得率和生产强度。本文对发酵过程中的培养基、通气条件、发酵动力学进行了研究。在此基础上,通过发酵过程中添加有机酸混合液和采取反复补料分批发酵的策略,显著提高了1,3-丙二醇的生产,具体实验结果如下：
利用正交实验,分别对种子培养基和发酵培养基进行了优化。在厌氧摇瓶中,利用优化后的发酵培养基,菌体生长和甘油的消耗显著提高,1,3-丙二醇的浓度和得率分别达到6.06 g·L-1和0.41 mol·mol-1,分别比在原始发酵培养基下提高了62%和7.9%。在5-L发酵罐中也得到类似的结果,并且优化后的发酵培养基中酵母提取物和氯化铵的浓度分别比原始发酵培养基显著下降,降低了生产的原料成本。
在原始发酵条件下,采取两阶段通气发酵及在发酵后期加入营养物质的策略不能提高1,3-丙二醇的生产。在250-mL摇瓶中,发现装液量增大时,有利于甘油的消耗和1,3-丙二醇的生成。在此基础上,分别研究了以甘油为唯一碳源情况下,不同通气方式对1,3-丙二醇生产的影响。结果显示,发酵过程中全程厌氧通气相比两阶段通气和通入空气,更有利于1,3-丙二醇的生产。
在补料分批发酵过程中,菌体的比生长速率随着产物的积累而不断的下降,进而影响到甘油的消耗和1,3-丙二醇的生成。在此基础上,通过甘油的限制性流加,控制菌体的比生长速率,发现在较低的菌体比生长速率下,甘油比消耗速率及其它产物的比生成速率较低,但是乙醇和甲酸的得率很高。随着菌体比生长速率的增加,甘油的比消耗速率和1,3-丙二醇的比生成速率迅速增加,同时乙醇和甲酸的得率迅速下降,1,3-丙二醇的得率大幅度增加。然后,在菌体生长和1,3-丙二醇生成明显减慢的发酵后期,通过控制不同的甘油浓度,发现甘油浓度在15-26 g.L-1时最有利于1,3-丙二醇的生成。最后,采取控制较高的菌体比生长速率和在发酵后期控制适宜甘油浓度相结合的策略,有效的减少了发酵过程中乳酸和乙醇的生成,提高1,3-丙二醇的得率。
在不对菌体进行基因工程操作的情况下,通过在发酵过程中外源添加有机酸混合液,有效的提高了1,3-丙二醇的生产,降低了乳酸和乙醇等副产物的生成。在此基础上,分别考察了不同浓度有机酸混合液加入时和在不同发酵时间加入时对发酵结果的影响。结果显示,在菌体浓度(OD650)达到3.0时,加入3.80 mM的有机酸混合液(柠檬酸、琥珀酸和延胡索酸均等浓度)最有利于1,3-丙二醇的生成,在30 h时达到了70 g.L-1以上。
在补料分批发酵过程中,产物积累到一定浓度时,就会对菌体生长产生很强的抑制。在厌氧摇瓶中通过反复分批培养,菌体生长和1,3-丙二醇的生产能力保持相对恒定；而在5-L发酵罐中发现,菌体长时间暴露在高浓度的产物中,不利于下一发酵循环的菌体生长。通过初始发酵条件下的反复补料分批发酵,1,3-丙二醇的生产强度为2.86g.L-1.h-1,但其浓度仅为44.60 g.L-1。在此基础上,采取反复补料分批发酵和有机酸添加相结合的策略,1,3-丙二醇生产有了显著的提高。在酵母浸膏代替酵母提取物的基础上,采取多循环的反复补料分批发酵操作,1,3-丙二醇的浓度、得率和生产强度在发酵20 h左右时分别达到66 g·L-1.0.61 mol·mol-1和3.40 g·L-1·h-1左右,显著的提高了1,3-丙二醇的生产。
1,3-propanediol has become one of the most interesting feedstocks in recent years because of its wide industrial applications, such as synthesis of polytrimethylene terephthalate and other polyester fibers. Current commercial production is based on the chemical route, which uses acrolein or ethylene as the starting material. Compared with the chemical synthesis method, biotransformation has the advantage of utilizing inexpensive, renewable resources such as glycerol or glucose, and becomes increasingly attractive.
To make the price more attractive, the yield, productivity and concentration of 1,3-propanediol need to be enhanced. In this paper, we had a study on culture medium optimization, aeration conditions and dynamics of fed-batch culture. Based on this, 1,3-propanediol production was enhanced by repeated fed-batch culture with organic acids addition, and the results of experiments were as follows.
The composition of medium for seeds culture and fermentation were optimized separately by orthogonal experiments. Based on this, the cell growth and glycerol consumption increased significantly in anaerobic bottle and the concentration and yield of 1,3-propanediol reached 6.06 g-L-'and 0.41 mol-mol-1, respectively,62% and 7.9% higher than that of the control, respectively. At the same time, the 1,3-propanediol production in 5-L bioreactor was also enhanced significantly, similar to that in anaerobic bottle. In addition, the concentrations of yeast extract and ammonium chloride both decreased sharply compared with that of the control, which could decrease the cost of the 1,3-propanediol production.
Under initial conditions, the 1,3-propanediol production would not be enhanced under the strategy of two-stage aeration and nutrient addition in late phase of fermentation. At the same time, experiments carried out in 250-mL with aerobic condition indicated that the increased volume of culture medium would be benefitial to PDO production. Based on this, effects of strategies including two-stagy aerating, aeration air and anaerobic aeration on the 1,3-propanediol production was studied separately as the glycerol was used as the solely carbon source, the results of experiments indicated that aerating N2 to maintain anaerobic condition would be benefitial to 1,3-propanediol production than that of others.
The specific growth rate of cells in fed-batch culture decreased as the products increasingly accumulated in culture medium, which further would not be benefitial to glycerol consumption and 1,3-propanediol production. Based on this, the specific growth rate was controlled by glycerol feeding rate. The experimental results indicated the specific growth rate affected significantly yields of productions. The yields of ethanol and formic acid on glycerol were high as the specific growth rate was maintained at low level. Increased the specific growth rate, the yields of ethanol and formic acid on glycerol decreased sharply while the yield of 1,3-propanediol on glycerol increased significantly. At the same time, the glycerol specific consumption rate and PDO formation rate both rapidly increased. As the cell growth and 1,3-propanediol specific formation rate declined sharply in late phase of fermentation, the glycerol concentration at the range of 15-26 g-L-1 was testified to be beneficial to the yield of 1,3-propanediol increase. Combined the specific growth rate controlled at higher level and glycerol concentration maintained range of 15-26 g-L-1 could effectively decreased the yields of lactic acid and ethanol on glycerol while increased the yield of 1,3-propanediol on glycerol.
The 1,3-propanediol production could be enhanced significantly while lactic acid and ethanol production decreased sharply with the parent strain under the strategy of organic acids mixture addition in the process of fermentation. Based on this, the effects of concentration of organic acids mixture addtion and time of organic acids mixture addition was studied separately and the results indicated that 3.80 mM organic acids mixture was added at the biomass (OD650) of 3.0 would be benefitial to 1,3-propanediol production and the concentration of 1,3-propanediol reached above 70 g-L"1at 30 h of culture.
The accumulation of products in medium of fed-batch culture would strongly inhibit cell growth. The strain used for 1,3-propanediol production could maintain the ability of 1,3-propanediol production in long-time cultivation in repeated batch culture in anaerobic bottle. However, when the cells used as inoculums which was maintained for a long-time in late phase of culture would not be benefitial to cell growth in next cycle of repeated fed-batch culture. The 1,3-propanediol productivity and concentration reached 2.86 g-L"--h-1and 44.60 g·LL1, respectively, in repeated fed-batch culture under initial condition. Based on this, the 1,3-propanediol production could be further enhanced in repeated fed-batch culture with organic acids mixture addition. At the same time, multiple-cycle of repeated fed-batch culture with industry-grade yeast extract replacing regent-grade yeast extract, the concentration, yield and productivity of 1,3-propanediol reached 66 g-L-,0.61 mol-mol1 and 3.40 g-L--h-, respectively, which was higher than that of the reported.
引文
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